Methods for the rapid detection and serotyping of pathogens are of high interest, due in part to the dramatic improvement in treatment efficacy for a bacterial or viral infection diagnosed early relative to one diagnosed at a later stage (Inglesby et al., “Anthrax as a Biological Weapon: Medical and Public Health Management,” J. Am. Med. Assoc. 281:1735-1745 (1999)). Unfortunately, most current methods of pathogen identification rely on some level of sample manipulation, (enrichment, fluorescent tagging, etc.) which can be costly in terms of both time and money. Thus, eliminating sample labeling will result in a significant savings and has the potential to speed diagnosis. The use of DNA hairpins as “molecular beacons” (Broude, “Stem-loop Oligonucleotides: a Robust Tool for Molecular Biology and Biotechnology,” Trends Biotechnol. 20:249-256 (2002)), either in solution (Tyagi et al., “Molecular Beacons: Probes that Fluoresce upon Hybridization,” Nature Biotech. 19:365-370 (2001); Dubertret et al., “Single-mismatch Detection Using Gold-quenched Fluorescent Oligonucleotides,” Nature Biotech. 19:365-370 (2001)) or immobilized on a solid surface (Fang et al., “Designing a Novel Molecular Bacon for Surface-Immobilized DNA Hybridization Studies,” J. Am. Chem. Soc. 121:2921-2922 (1999); Wang et al., “Label Free Hybridization Detection of Single Nucleotide Mismatch by Immobilization of Molecular Beacons on Agorose Film,” Nucl. Acids. Res. 30:61 (2002); Du et al., “Hybridization-based Unquenching of DNA Hairpins on Au Surfaces: Prototypical “Molecular Beacon” Biosensors,” J. Am. Chem. Soc. 125:4012:4013 (2003); Fan et al., “Electrochemical Interrogation of Conformational Changes as a Reagentless Method for the Sequence-specific Detection of DNA,” Proc. Natl. Acad. Sci. USA 100:9134-9137 (2003)), has proven to be a useful method for “label-free” detection of oligonucleotides. Molecular beacons consist of DNA hairpins functionalized at one terminus with a fluorophore and at the other terminus with a quencher. In the absence of their complement, they exist in a closed, “dark” conformation. Hybridization occurs on introduction of complementary oligonucleotides, which concomitantly forces open the hairpin and allows for a fluorescent, “bright” state.
Traditionally, as illustrated in FIG. 1, molecular beacons have been designed by supplementing the targeted DNA sequence at both termini with additional self-complementary nucleotides to force the formation of a hairpin (Monre et al., “Molecular Beacon Sequence Design Algorithm,” Biotechniques 34:68-73 (2003)). While generally successful, the addition of non target-derived nucleotides increases the potential for non-specific binding, thus potentially reducing both the sensitivity and selectivity of the probe beacon. Modifications of this discovery protocol, such as the “shared stem” methodology of Bao and coworkers (Tsourkas et al., “Structure-function Relationships of Shared-Stem and Conventional Molecular Beacons,” Nucl. Acids Res. 30:4208-4215 (2002)), still incorporate several bases unrelated to the target sequence. Thus, the latter approach potentially suffers from the same deficiencies. It would be desirable to identify a reliable approach for identifying DNA hairpins that overcomes the above-noted deficiencies.
The present invention is directed to achieving these objectives and otherwise overcoming the above-noted deficiencies in the art.